133. Use of 2As To Control Protein Subcellular Localization

Abstract

A substantial proportion (~39%) of all human proteins are either secreted from the cell, located within the lumen/ membranes of cytoplasmic vesicular structures, or, are plasma membrane proteins. Given that such high proportion of proteins are initially translocated into the endoplasmic reticulum (ER), many therapeutic strategies rely on the ability to co-express multiple proteins – some, or all of which, might be targeted to such sites. This directly applies to in vivo gene therapy strategies, or, when therapeutic proteins may need to be co-expressed with selectable markers (e.g. ex vivo gene therapies).As was shown before, Picornavirus 2A (foot-and-mouth disease virus 2A; F2A) and ‘2A-like’ sequences are powerful tools that allow multiple proteins to be translated and co-expressed from a single transcript mRNA under the control of only one promoter. When 2A is positioned between sequences encoding two, or more, genes, it mediates a co-translational ‘cleavage’ at its own C-terminus. A major problem with co-expression of certain proteins targeted to, or transiting through, the ER is that the ‘cleavage’ activity of short F2As can be greatly inhibited by sequences immediately upstream leading to aberrant sub-cellular localisation of some proteins.We have also discovered a number of active cellular 2A-like sequences, associated with non-long terminal repeat (non-LTR) retrotransposons, but also with structural and metabolic proteins: ankyrin repeats, sodium dependent neutral amino acid transporters, and NOD-like receptor (NLR) proteins. Examination of the surrounding protein and gene structure revealed that in the majority of cases these 2A sequences occurred as N-terminal features. Interestingly, using SignalP, many of these novel 2As scored highly as N-terminal signal peptides.Here, we present our latest findings on 2A sequences. A series of test proteins (eGFP and mCherry) were expressed i) followed by ‘hybrid’ ‘self-cleaving’ F2A sequences (with different upstream contexts) or ii) downstream of a putative signal 2A. We demonstrate that inhibition of F2A-mediated cleavage in shorter sequences can be overcome by introduction of mutations upstream of 2A changing the context of the sequence between the C-terminus of the upstream protein and 2A sequence. In the case of N-terminal – cellular – (NLR) 2As, ‘uncleaved’ 2A indeed can act as a signal peptide. If 2A does not ‘cleave’, it directs a proportion of the newly synthesised reporter protein to the exocytic pathway: if 2A ‘cleaves’, the protein downstream is localised to the cytoplasm. This type of 2A mediates, therefore, a newly discovered form of dual protein targeting.